I LECTURE 5: AFM IMAGING - MIT OpenCourseWare€¦ · AFM can be combined with high resolution...

3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

I

LECTURE 5: AFM IMAGING

Outline : LAST TIME : HRFS AND FORCE-DISTANCE CURVES .......................................................................... 2 ATOMIC FORCE MICROSCOPY : GENERAL COMPONENTS AND FUNCTIONS................................. 3 Deflection vs. Height Images ................................................................................................... 4 3D Plots and 2D Section Profiles ............................................................................................ 5 Normal Modes of Operation ..................................................................................................... 6 Other Modes of Operation........................................................................................................ 7 Factors Affecting Resolution .................................................................................................... 8 Tip Deconvolution .................................................................................................................... 9 Imaging of Cells ..................................................................................................................... 10 Imaging of DNA...................................................................................................................... 11 HRFS COMBINED WITH AFM : SPATIALLY SPECIFIC SURFACE INTERACTIONS........................... 12

Objectives: To review the basic principles, capabilities, and current state of the art uses of the atomic force microscopy

Readings: Course Reader Document 12-15.

Multimedia : Watch Introduction to AFM by Asylum Research, Inc. (Quicktime Movie) for Lectures 4-5.

1


HIGH RESOLUTION FORCE SPECTROSCOPY (HRFS) EXPERIMENT : FORCE-DISTANCE CURVES

approaching

retracting

A.tip and sampleout of contact

B.attractive

interaction pulls tip down

towards surface

δ1=0

δ2<0

δ3<0

δ4>0

C.tip jumpsto surface

D.tip and sample

/ z-piezomove in unison

E.attractive force

keeps tip incontact with

surface

F.tip releasesfrom surface

G.tip and sampleout of contact

(δ=cantilever deflection)

D.tip and sample

/ z-piezomove in unison

-Normal vs. Lateral Force Microscopy

Tip-Sample Separation Distance, D (nm)

Forc

e, F

(nN

)

repulsiveregime

attractive regime

0

kc

- Conversion of raw data; sensor output, s (Volts) vs. z-piezo displacement/deflection, δ (nm) to Force, F, versus tip-sample separation distance, D : δ=s/m m= slope in constant compliance regime =Δs/Δδ (V/nm) F=kδ D= z±δ -zeroing the baseline

2


ATOMIC FORCE MICROSCOPY : GENERAL COMPONENTS AND FUNCTIONS

sample

sensor output, δ, Flaser diode

feedback loop• controls z-sample

position

position sensitive photodetector• measures deflection of

cantilever

mirror A BC D

ERROR = actual signal - set point

δ

cantileverhich deflects as

≈10°-15°

• spring w probe tip scans sample surface

computer • controls system

xyz

• performs data acquisition,display, and analysis

piezoelectric scannerpositions samp

probe tip• senses surface

• le (x, y, z) with Å

accuracy

properties and causescantilever to deflect

Advantages : 1) Unlike electron microscopes, samples do not need to be coated or stained, minimal damage, 2) Unlike electron microscopes, samples can be imaged in fluid environments (near-physiological conditions), 3) Unlike STM samples do not need to be conductive, 4) Sub-nm resolutions have been achieved on biological samples (detailed information on the molecular conformation, spatial arrangement, structural dimensions, rate dependent processes, etc.)

-Piezo rasters or scans in the x-y direction across the sample surface

↓

-Cantilever deflects (δ) in response to an a topographical feature (hill or valley)

↓ Feedback loop

-System continuously changes in response to an experimental output (δ= cantilever deflection which is the feedback parameter)

-Computer adjusts the piezo z-displacement to keep δ constant = "setpoint"

↓

Error signal (actual signal-set point) used to produce a topographical (height) map in the z-direction of the surface

3


ATOMIC FORCE MICROSCOPY : DEFLECTION VS HEIGHT IMAGES

Two images removed due to copyright restrictions. See Tai and Ortiz, Nano Letters 2006.

Deflection Image: -Raw data output of cantilever deflection from

photodiode, very clear, the less feedback the clearer, One can identify and measure high resolution (x/y)

spatial dimensions of structural features

Height Image: -Processed data from z-piezo, less clear compared to deflection image, maximize your feedback system, can quantify the height of structural features, in 2D image corresponds to brightness

(e.g. images are a large residual nanoindent of bone using an instrumented indenter and Berkovich diamond probe showing plastic deformation of mineralite nanogranular structure, K. Tai and C. Ortiz Nano Letters, 2006)

4


ATOMIC FORCE MICROSCOPY : 3D PLOTS AND 2D SECTION PROFILES

Two images removed due to copyright restrictions. See Tai and Ortiz, Nano Letters 2006 3D Height image 2D Height image

X[µm]

76543210

7

6

5

4

3

2

Z[Å

]

1

0

z (μm)

x (μm)

1.5 1.0 0.5 0

0 2 4 6 8 10

2D Section Profile

-Select linear region of plot and plot 2D section profile (height along line) z vs. x to get quantitative mathematical functional form of topography. For example, we can see the profile of the deformation of indent plus form of plastically deformed "pileup" regions"→ one can use these profiles in conjunction with modeling to extract out material properties such as modulus, yield stress, anisotropy, and strain hardening behavior. -In the next assignment, you will have to use a section profile on nanoparticles to estimate the probe tip radius. (e.g. images are a large residual nanoindent of bone using an instrumented indenter and Berkovich diamond probe showing plastic deformation of mineralite nanogranular structure, K. Tai and C. Ortiz Nano Letters, 2006)

5


ATOMIC FORCE MICROSCOPY IMAGING : NORMAL MODES OF OPERATION

Force

Intermittent-contact

contactDistance (tip-to-sample separation)

non-contact

Graph by MIT OCW. Schematic after Thermomicroscopes "Introduction to AFM."

Contact (DC and AC or Force Modulation) : ● tip remains in contact with sample ● feedback signal, δc ● potentially destructive due to high lateral (x/y) forces ● high resolution Intermittent Contact (AC, Tapping) Mode : ● tip is oscillated near its resonant frequency and "taps" on and off the surface ● feedback signal, oscillation amplitude or phase ● less destructive due to reduction of lateral forces ● loss of spatial resolution Noncontact (AC) Mode : ● tip is oscillated near its resonant frequency without touching the surface ● feedback signal, oscillation amplitude ● nondestructive ● loss of spatial resolution ● difficult in practice

6


ATOMIC FORCE MICROSCOPY IMAGING : OTHER MODES OF OPERATION

chemically functionalizedprobe tip:

X=-OH,-CH3,-NH2

XX

XX

XX

XXXXXXXXX

XX

XX

Xchemically functionalized

probe tip: X=-OH,-CH3,-NH2

XX

XX

XX

XXXXXXXXX

XX

XX

X

IV. Chemical Force Microscopy (CFM)

Frisbie, et al., 1994

II. Friction or Lateral Force Microscopy (FFM/ LFM)

I. Normal Force Microscopy

III. Force / Volume Adhesion Microscopy

Radmacher, et al., 1994

Noncontact (NC) 1995

Frisbie, et al., 1994

Contact DC and AC (Force Modulation

Microscopy (FMM), Phase Imaging) Hansma, et al., 1991

Intermittant Contact/ Tapping / Lift (AC)Hansma, et al., 1994

http://www.di.com/AppNotes/ForceVol/FV.array.html

http://www.di.com/AppNotes/LatChem/LatChemMain.html

Surface Maps:Topography & Rough c Interactions, Friction

Chemical, Adhesion , Hardness, Elasticity /Viscoelasticityness, Electrostati

Dynamic Processes :Erosion, De Interactionsgradation, Protein-DNA

7

Courtesy of Veeco Instruments. Used with permission.

Courtesy of Veeco Instruments. Used with permission.


ATOMIC FORCE MICROSCOPY IMAGING : FACTORS AFFECTING RESOLUTION

voltageapplied

L

L+ΔL

electrodes

connecting wires

d

+Y -X +X

D

D+ΔD

polarization

x

yz

+Z

-Z

~

PROBE TIP SHARPNESSCANTILEVER

THERMAL NOISESheng, et al. J. Microscopy 1999, 196, 1.

Lindsay Scanning Tunneling Microscopy and Spectroscopy 1993, 335. ktao, et al. Ultramicroscopy 1996,

66, 141. =m

cantilever δt(max) m

δt(max) m

Sh

Distance, D (nm)

Forc

e, F

(nN

)

0

Fadhesion

0

PIEZO AMPLIFIER, SENSOR AND CONTROL ELECTRONICS,

MECHANICAL PARAMETERS

SPECIMEN DEFORMATION &

THERMAL FLUCTUATIONS

ADHESION FORCE

Physik Instruments, Nanopositioning 1998

Hoh, et al. Biophys. J. 1998, 75, 1076.

Yang, et al. Ultramicroscopy 1993, 50, 157

8

Image removed due to copyright restrictions. Image removed due to copyright restrictions.

3-D model of sharp probe tip on a protein,from Lieber et al, 2000 (http://cnst.rice.edu)


ATOMIC FORCE MICROSCOPY IMAGING : TIP DECONVOLUTION -Imaging very sharp vertical surfaces is influenced by the sharpness of the tip. Only a tip with sufficient sharpness can properly image a given z-gradient. Some gradients will be steeper or sharper than any tip can be expected to image without artifact. False images are generated that reflect the self-image of the tip surface, rather than the object surface. Mathematical methods of tip deconvolution can be employed for image restoration. The effectiveness of these methods will depend on the specific characteristics of the sample and the probe tip.

9


10

ATOMIC FORCE MICROSCOPY IMAGING OF CELLS Contact mode image of human red blood cells - note cytoskeleton

is visible. blood obtained from Johathan Ashmore, Professor of Physiology University College, London. A false color table has been used here, as professorial blood is in fact blue. 15µm scan courtesy

M. Miles and J. Ashmore, University of Bristol, U.K.

Rat Embryo Fibroblast (*M. Stolz,C. Schoenenberger, M.E. Müller Institute,

Biozentrum, Basel Switzerland)

Height image of endothelial cells taking in fluid using Contact Mode AFM. 65 µm scan courtesy J. Struckmeier, S. Hohlbauch, P. Fowler, Digital Intruments/Veeco Metrology, Santa Barbara, USA.

Red Blood CellsShao, et al., : http://www.people.virginia.edu/~js6s/zsfig/random.html

Radmacher, et al., Cardiac Cellshttp://www.physik3.gwdg.de/~radmacher/

Courtesy of Manfred Radmacher. Used withpermission.

Courtesy of Mervyn J. Miles. Used withpermission.


ATOMIC FORCE MICROSCOPY IMAGING OF DNA Image of PtyrTlac supercoiled DNA. 750 nm scan courtesy C. Tolksdorf, Digital Instruments/Veeco,

Santa Barbara, USA, and R. Schneider and G. Muskhelishvili, Istitut für Genetik und

Mikrobiologie, Germany.

Tapping Mode image of nucleosomal DNA.Courtesy of Yuri Lyubchenko. Used with permission.

The high resolution of the SPM is able to

discern very subtle features such as these two linear dsDNA molecules overlapping

each other. 155nm scan.

11

Curtesy of Veeco Instruments andG. Muskhelishvili. Used with permission.

Courtesy of Zhifeng Shao. Used with permission.

Courtesy of Prof. Carlos Bustamante. Used with permission.

http://people.virginia.edu/~zs9q/zsfig/DNA.html

Courtesy of W. Blaine Stine. Used with permission.

Courtesy of Zhifeng Shao. Used with permission.

AFM image of short DNA fragment with RNA

polymerase molecule bound to transcriptionrecognition site. 238 nm size. Courtesy

of Bustamante Lab. Chemistry Department.University of Oregon, Europe OR


HRFS COMBINED WITH AFM : SPATIALLY SPECIFIC SURFACES INTERACTION INFORMATION

1 μm

Grain 1

Grain 2 Grain 3

12

34 5 6

7

1 μm

Grain 1

Grain 2 Grain 3

12

34 5 6

7

1 μm1 μm

Grain 1

Grain 2 Grain 3

12

34 5 6

7

12

34 5 6

7

0.0

0.1

0.2

0.3

0.4

0 5 10 15Distance from Surface (nm)

Forc

e (n

N)

0

1

2

3

4 Force/Radius (m

N/m

)

Position 1Position 2Position 3Position 4Position 5Position 6Position 7

● AFM can be combined with high resolution force spectroscopy and nanoindentation since cantilever probe tip can be employed for both imaging and nanomechanical measurements→ nanomechanical measurements with positional accuracy down to the nanoscale (Vandiver, et al. Biomaterials 26 (2005) 271–283).

12

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

I LECTURE 5: AFM IMAGING - MIT OpenCourseWare€¦ · AFM can be combined with high resolution...

Documents

Transcript of I LECTURE 5: AFM IMAGING - MIT OpenCourseWare€¦ · AFM can be combined with high resolution...